A protein kinase is an enzyme which catalyzes phosphorylation of hydroxyl groups on tyrosine, serine and threonine residues of proteins. It plays an important role in signal transduction of growth factors involved in growth, differentiation and proliferation of cells.
For maintenance of homeostasis, it is essential that turning on and off of the signal transduction system be well balanced. However, mutation or overexpression of specific protein kinases disrupts the signal transduction system in normal cells (a state when in vivo signal transduction is continuously turned on) and causes various kinds of diseases including cancers, inflammations, metabolic diseases, brain diseases, or the like. Typical protein kinases that lead to diseases caused by abnormal cell growth include Raf, KDR, Fms, Tie2, SAPK2a, Ret, Abl, Abl(T315I), ALK, Aurora A, Bmx, CDK/cyclinE, Kit, Src, EGFR, EphA1, FGFR3, Flt3, Fms, IGF-1R, IKKb, IR, Itk, JAK2, KDR, Met, mTOR, PDGFRa, Plk1, Ret, Syk, Tie2, TrtB, and so forth.
Human genome is believed to contain 518 protein kinase genes and they constitute about 1.7% of all human genes [Manning et al., Science, 2002, 298, 1912]. Human protein kinases are largely divided into tyrosine-specific protein kinases (over 90 species) and serine/threonine-specific protein kinases. The tyrosine-specific protein kinases may be divided into 58 receptor tyrosine kinases, which are again grouped into 20 subfamilies, and 32 cytoplasmic/non-receptor tyrosine kinases, which are grouped into 10 subfamilies. The receptor tyrosine kinase has an extracellular domain capable of binding to a growth factor and a cytoplasmic active site that can phosphorylate the tyrosine residue. When a growth factor binds to the extracellular growth factor receptor site of the receptor tyrosine kinase, the receptor tyrosine kinase forms a dimer and the tyrosine residues in the cytoplasm are autophosphorylated. Then, the downstream proteins are sequentially phosphorylated, and as the signal transduction proceeds in the nucleus, the transcription factors that induce cancer are overexpressed in the end.
Raf is a serine/threonine (Ser/Thr)-specific protein kinase and serves the role of transmitting biological signals from activated growth factor receptors on the cell membrane into the nucleus. The mitogen-activated protein kinase (MAPK) signal transduction system is essential in cellular proliferation, division, survival, apoptosis, and the like. The MAPK signal transduction system largely consists of three kinase phosphorylation processes—i.e., sequential phosphorylation of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. Raf is a MAPKKK, MEK is a MAPKK, and the extracellular signal-regulated kinase (ERK) is a MAPK. When the receptor is activated, the small GTP-binding protein, Ras, is activated and the MAPK signal transduction into the nucleus is performed through sequential phosphorylation of Raf-MEK-ERK.
The Ras oncogene (especially k-Ras) in a permanently activated state is closely related to the onset of solid tumors such as pancreatic cancer (about 90%), rectal cancer (about 45%), liver cancer (about 30%), non-small cell lung cancer (about 35%), renal cancer (about 10%), or the like. If Raf-1 binds to activated Ras, serine 338 of Raf-1 is phosphorylated [Avruch, J. Recent Progress in Hormone Research, 2001, 56, 127], and the Raf-1 is activated. In contrast, if 14-3-3 protein binds to Raf-1 with phosphorylated serine 259, the Raf-1 is inactivated. The Raf kinase is also involved in the nuclear factor-κB (NF-κB) signal transduction system, which plays a key role in immune responses and inflammations [Caraglia, M. et al., Annals of Oncology, 2006, 17, 124]. That is, Raf phosphorylates inactivated IκB protein and induces migration of NF-κB protein into the nucleus, thereby stimulating a transcription factor that inhibits apoptosis.
Another apoptosis inhibition mechanism of Raf is as follows. Raf forms a dimer together with Bcl-2 and is translocated into the mitochondria. There, it phosphorylates Bad, thereby initiating apoptosis inhibition by Bcl-2. Accordingly, Raf is immunoprecipitated along with Bcl-2 [Yuryev, A. et al., Mol. Cell. Biol., 2000, 20, 4870].
The three subtypes of Raf protein (A-Raf, B-Raf and C-Raf/Raf-1) have three conserved regions (CR1, CR2 and CR3) at the N-terminal regulatory domain and the C-terminal kinase domain. CR1 includes a Ras-binding domain (RBD) such as the cysteine-rich domain (CRD), CR2 includes a 14-3-3 protein-binding site (e.g., serine 259 of Raf-1), and CR3 includes a catalytic domain [Tran et al., J. Biol. Chem., 2005, 280, 16244] and two activation segment phosphorylation sites (threonine 491 and serine 494 of Raf-1) [Wellbrock, C. Nature Reviews Molecular Cell Biology, 2004, 5, 875]. The three subtypes of Raf protein are expressed in different tissues. Whereas C-Raf is expressed in almost all tissues, A-Raf is mainly expressed in urogenital tissues (e.g., kidney, uterus and prostate gland) and B-Raf is mainly expressed in nervous, splenic and hematopoietic tissues [Jaiswal, R. K. et al., J. Biol. Chem., 1966, 271, 23626].
Mutation of B-Raf is known to be associated with about 7% of all human cancers. Especially, it has been observed with high frequency (˜70%) in melanoma, a type of skin cancer. Of the mutations of B-Raf, the B-Raf-V600E mutation, i.e., a point mutation with valine 600 of exon 15 being replaced by glutamic acid, mainly (about 90%) induce melanoma [Davies, H. et al., Nature 2002, 417, 949]. As compared with wild-type B-Raf, B-Raf-V600E has about 500 times higher in vitro kinase activity. Accordingly, B-Raf-V600E induces hyperactivation of the MAPK signal transduction and leads to cancer. The reason why B-Raf-V600E has such a high kinase activity is as follows. The glutamic acid 600 replaced by the point mutation mimics a phosphate group between the phosphorylation sites (threonine 598 and serine 601) located at the activation segment and, thereby, induces structural conformation of the permanently activated B-Raf kinase domain [Tuveson, D. A., Cancer Cell, 2003, 4, 95]. Up to the present, about 40 B-Raf mutations were found (mainly at the activation segment and the glycine-rich G-loop of the catalytic domain). However, occurrence of mutations other than V600E is fairly infrequent. In rectal cancer, about 10% of B-Raf mutations occur at the G-loop of the catalytic domain [Rajagopalan et al., Nature 2002 418, 934].
Although B-Raf has an auto-inhibition domain at the N-terminal, B-Raf becomes permanently activated when activated H-Ras binds thereto. This is caused by phosphorylation of serine 445. The phosphorylation of serine 338 of C-Raf corresponds to that of serine 445 of B-Raf. The B-Raf V600E mutation inhibits the auto-inhibition mechanism of B-Raf and turns it permanently activated.
The B-Raf-V600E mutation is observed at high frequency (about 50%) in papillary thyroid cancer [Salvatore, G. J. Clin. Endocrinol. Metab. 2004, 89, 5175]. Also, the B-Raf-V600E is closely associated with the onset of colon cancer (about 20%) and uterine cancer (about 30%).
Also, hyperactivation of C-Raf without oncogenic mutation is observed in renal carcinoma (about 50%) and hepatocellular carcinoma (HCC) (about 100%).
Sorafenib (BAY 43-9006, marketed as Nexavar) developed by Bayer and Onyx strongly inhibits C-Raf and both wild-type and mutant B-Raf. Further, sorafenib inhibits activity of the receptor tyrosine kinases, such as platelet-derived growth factor receptor, vascular endothelial growth factor receptors 1/2/3, fibroblast growth factor receptor, Flt-3, c-Kit, RET, or the like. It inhibits the kinase by stabilizing the DGF motif of the kinase domain to have an inactive conformation [Wan, P. T. et al., Cell, 2004, 116, 855]. Sorafenib was approved as a treatment for advanced renal cell carcinoma in 2005. The therapeutic effect of sorafenib on renal cancer originates from to the inhibition of vascular endothelial growth factor receptors 1/2/3 and other kinases rather than the inhibition of Raf. In the clinical trial phase II, a maximum allowed administration dose of sorafenib was 400 mg (twice a day). Administration of 600 mg (twice a day) of sorafenib may lead to grade 3 skin toxicity. Frequent adverse effects of sorafenib include hand-foot syndromes such as peeling of skin, rash and edema. In 2008, sorafenib was approved as a treatment for hepatocellular carcinoma. In addition, sorafenib showed therapeutic effect for intractable thyroid cancer, hormone-refractory prostate cancer and breast cancer in a clinical trial phase II. However, sorafenib shows no therapeutic effect on the skin cancer melanoma.
PLX4720, a 7-azaindole derivative developed by Plexxikon, induces apoptosis of melanoma cells such as 1205Lu (Raf-V660E overexpressed cells) [Tsai, J. et. al., PNAS, 2008, 105, 3041]. PLX4720 is a potent inhibitor of Raf-V660E kinase activity (IC50=13 nM) and also inhibits the proliferation of A375 melanoma cells (IC50=0.5 μM).
CHIR265 developed by Novartis and Chiron also strongly inhibits the kinase activity of B-Raf-V600E (IC50=19 nM), KDR (IC50=70 nM), PDGFR-b (IC50=30 nM) and c-Kit (IC50=20 nM). CHIR265 is currently in clinical trial phase I for melanoma patients.
Resistance to Raf inhibitors has been an emerging issue. Montagut et al. explained the mechanism of resistance to the Raf inhibitor by culturing M14 cells (human melanoma cells) with B-Raf-V600E mutation in the presence of a Raf inhibitor (AZ628) and acquiring clones resistant to the Raf inhibitor. Inhibition of B-Raf results in increased expression of C-Raf protein and decreased inhibitory effect on B-Raf-V600E. Meanwhile, the melanoma cells resistant to the Raf inhibitor (AZ628) exhibit increased susceptibility to the HSP90 inhibitor geldanamycin. Thus, inhibition of HSP90 may be a way to overcome the resistance to the Raf inhibitor [Montagut, C. Cancer Research, 2008, 68, 4853]:
Vascular endothelial growth factor receptors (VEGFRs) are receptor tyrosine kinases (RTKs) and important regulatory factors of angiogenesis. They are involved in the formation of blood vessels and lymphatic vessels and in homeostasis, and exert important effects on nerve cell. Vascular endothelial growth factor (VEGF) is produced mostly by vascular endothelial cells, hematopoietic cells and stromal cells under a hypoxic condition or by stimulations from growth factors such as TGF, interleukin and PDGF. VEGF binds to VEGFR-1, -2 and -3. Each VEGF isoform binds to a specific receptor, thereby inducing the formation of a receptor homozygote or heterozygote, and activates the corresponding signal transduction system. The signal specificity of VEGFR is further fine-tuned by co-receptors such as neuropilin, heparan sulfate, integrin, cadherin, or the like.
The biological function of VEGF is mediated by type III RTK, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4). VEGFR is closely related to Fms, Kit and PDGFR. Each VEGF binds to specific receptors. VEGF-A binds to VEGFR-1, -2 and receptor zygote, whereas VEGF-C binds to VEGF-2, -3. PIGF and VEGF-B interact exclusively with VEGFR-1, and VEGF-E interacts only with VEGFR-2. VEGF-F interacts with VEGFR-1 or -2. Whereas VEGF-A, -B and PIGF are preferentially required for the formation of blood vessels, VEGF-C and -D are essential in the formation of lymphatic vessels. Angiogenesis is essential in the proliferation and transition of tumors, since it supplies nutrients and oxygen to the tumors and helps transition to cancer cells. Normally, angiogenesis is balanced by angiogenic stimulators and angiogenic inhibitors. If the balance is broken, as in cancer cells, the growth factor that affects the vascular endothelial cells most, i.e., VEGF, activates its receptor, VEGFR. At present, various researches are under way on the inhibitors that inhibit the receptor tyrosine kinase of VEGF using low-molecular-weight synthetic substances, which are advantageous in that they are applicable also to solid tumors and have fewer side effects because they inhibit angiogenesis in cancer cells only.
Tie2 is a kind of receptor tyrosine kinase which is deeply involved with angiogenesis and vasculature. The Tie2 domain structure is highly conserved in all vertebrates [Lyons et al., 1998]. The ligand of Tie2 is angiopoietin (Ang). Ang2 does not induce autophosphorylation of Tie2, but interferes with the activation of Tie2 by Ang1. In endothelial cells, the activation of Tie2 by Ang2 induces activation of PI3K-Akt [Jones et al., 1999]. In the mitogen-activated protein kinase (MAPK) signal transduction pathway, which is the main signal transduction system of Tie2, the adaptor protein GRB2 and the protein tyrosine phosphatase SHP2 play a key role in dimerization of the Tie2 receptor tyrosine kinase through autophosphorylation. Ang/Tie2 and the VEGF signal transduction pathway are important in angiogenesis of cancer cells. Tie2 is expressed in vascular endothelial cells. Especially, the expression increases remarkably at the site invaded by cancer cells. Overexpression of Tie2 was observed in breast cancer [Peters et al., 1998] and also in uterine cancer, liver cancer and brain cancer.
Several compounds with an indole structure have been synthesized. However, the indole compound of the present invention with specific substituents at the 1-, 3- and 6-positions of indole has never been synthesized. Thus, of course, the inhibitory activity against various protein kinases or the possibility as an agent for treatment and prevention of cancers of the 1,3,6-substituted indole compound has never been disclosed in any literature.